Therapeutic antibodies (mAbs) have important applications in medicine. However, antibodies of non-human origin such as murine antibodies have drawbacks in that humans mount an anti-mouse antibody response when administered murine antibodies. Techniques developed to overcome the risk of immunogenicity involved increasing the content of human sequences in antibodies, including grafting murine complementarity-determining regions (CDRs) onto human frameworks (FRs) to create an antibody with higher human sequence content. This process, known as CDR-grafting (Jones, et al., 1986, Nature 321:522-525), was the first described method of antibody humanization. Since then, several methods of humanization have been described including resurfacing (Roguska, et al., 1994, Proc Natl Acad Sci USA 91:969-973), specificity-determining residue (SDR) grafting (Kashmiri, et al., 2005, Methods 36:25-34), superhumanization (Hwang, et al., 2005, Methods 36:35-42), human string content optimization (Lazar, et al., 2007, Mol Immunol 44:1986-1998), and framework shuffling (Dall'Acqua, et al., 2005, Methods 36:43-60; Damschroder, et al., 2007, Mol Immunol 44:3049-3060). The underlying assumption of all these methods is that the greater global sequence identity of the humanized sequence to a natural human sequence results in a lower risk of immunogenicity. However, due to the perceived risk of losing antigen affinity, none of these methods substantially engineer the CDRs, and as such none of these humanization methods reach the global sequence identity levels of human antibodies as they still contain mostly non-human CDRs. More recently, “fully-human” mAbs generated from recombinant human antibody libraries (Griffiths, et al., 1994, Embo J 13:3245-3260; Knappik, et al., 2000, J Mol Biol 296:57-86) or transgenic mice comprising human germline configuration immunoglobulin gene sequences (Lonberg, 2005, Nat Biotechnol 23:1117-1125; Green, et al., 1994, Nat Genet 7:13-21; Lonberg, et al., 1994, Nature 368:856-859) have emerged as alternatives to murine generated and subsequently humanized mAbs. These mAbs have both high affinity as well as high human sequence content.
Linking antibodies to moieties of interest such as drugs, radioactive elements, labels or stabilizing molecules (e.g. polymers) is of high pharmaceutical interest. Current methods used for conjugation to human or humanized antibodies rely solely on direct, non-enzymatically-mediated, functionalization of certain residues with moieties of interest. Human or humanized antibodies are functionalized on cysteine residues. However a major drawback of such methods is that conjugation is not stoichiometric, with antibodies in a mixture typically having from 1 to 8 moieties (e.g. drugs) per antibody, and that conjugation can occur on unwanted cysteine residues necessitating cysteine engineering. Enzymatic methods have been shown to be capable of conjugating moieties of interest onto murine antibodies. Notably, transglutaminases (TGases) have been used to conjugate small detectable markers onto antibodies having murine variable regions (see, e.g., Josten et al. (2000) J. Immunol Methods 240, 47-54; Mindt et al (2008) Bioconjug. Chem. 19, 271-278; Jeger et al (2010) Angew. Chem. Int. Ed. 49: 9995-9997); Kamiya et al (2003) Enzyme. Microb. Technol. 33, 492-496 and US patent publication no. 2011/0184147. However, the rules which govern selection by TGases of glutamine residues for modification are still largely unknown. Antibodies are estimated to have in the range of 90 lysine residues and 60 glutamine residues, including surface-exposed residues in the CDRs and FRs sequences. Current methods for producing antibody-drug conjugates are based on chemical reactions between naturally-occurring cysteines within antibodies and maleimide-reactive groups on drug-containing linkers. However such conjugation onto naturally occurring cysteine residues risks of unwanted conjugation onto different cysteines.
In view of the foregoing, there remains a need in the art for methods to conjugate moieties onto antibodies in a stoichiometric fashion and without negatively affecting the biological activity of the antibodies.